This project is a fundamental study examining the molecular-level processing and receptor interactions of peptides produced from the cocaine- and amphetamine-regulated transcript (CART), which plays important roles in glucose regulation and incretin secretion and may serve as a novel target for several diseases, including type 2 diabetes. However, progress in pursuing CART peptide signaling as a therapeutic target has been limited due to: ) the lack of rigorous evaluation of the post-translational processing of CART peptides across tissues; and 2) no receptor for CART peptides has been identified in any tissue. Based on strong literature evidence, we hypothesize that CART peptides are processed differently across peripheral tissues (including with uncharacterized post-translational modifications) and that there exist CART-specific cell-surface receptors responsible for facilitating their known biological functions. Here, we test these hypotheses through a combination of novel mass spectrometry (MS) and chemical biology approaches. Because the majority of studies on CART signaling focus on only one peptide form in the brain, our studies are innovative in that they rigorously evaluate additional molecular forms of CART peptides, do so in peripheral tissues associated with glucose regulation (in contrast to most studies, which focus on effects in the brain), and utilize novel techniques to identify the first receptor for CART peptides. The long-term objective of this research is to gain new insight into the molecular-level details of CART signaling to aid in the evaluation of CART signaling as a therapeutic target for disorders involved in glucose regulation. In Aim 1, we will develop a targeted MS assay to identify and quantify the final processed forms of CART peptides and use this assay to rigorously compare the relative processing of CART peptides across several cell types. To gain insight into the relevance of these forms for glucose homeostasis, we will evaluate the ability of each of these peptide forms to activate signaling pathways identified for CART in a model pancreatic beta cell line. In Aim 2, we will develop a novel MS-based technology, function-driven enrichment, to identify peptide receptor(s) that overcomes the limitations of many previous approaches to identify peptide-receptor interactions. We will apply this approach to identify the CART peptide receptor(s) present on the surface of model cells, which will be the first CART peptide receptor identified. Importantly, the function-driven enrichment approach developed here is not limited to determining the receptor for CART, and can be generalized to identify the receptors for other biologically active hormones and neuropeptides of interest in future studies. Overall, this research will significantly advance our understanding of cell-cell signaling in several different cell types, and may establish CART signaling as a novel therapeutic target.